Stabilisation of plant membranes during freezing by specific proteins

D.K. Hincha, B. Neukamm, F. Sieg and J.M. Schmitt,
Pflanzenphysiologie, Freie Universität Berlin, Germany

We have developed in vitro-assays using isolated spinach thylakoid membranes, to assess the cryoprotective activity of proteins. When thylakoids from nonacclimated plants are frozen and thawed under damaging conditions, they rupture and collapse. Collapsed thylakoid membranes can be distinguished from intact membranes by a volumetric hematocrit centrifugation assay and by immunological determination of the loss of the lumenal protein plastocyanin. We have isolated a cryoprotective protein (cryoprotectin) from cold acclimated cabbage (Brassica oleracea) leaves. Analysis by SDS-PAGE and silver staining revealed a single polypeptide of 7 kD in the most active fractions. Gel filtration chromatography confirmed that the cryoprotective activity is associated with a protein of 7 kD under nondenaturing conditions. This protein could be isolated only from cold acclimated leaves, but not from plants grown under nonacclimating conditions. N-terminal sequencing and comparison with sequence data banks revealed a high degree of sequence identity with several plant lipid transfer proteins, especially with the family of WAX proteins from Brassica. An isolated WAX protein from the cuticle of cabbage leaves had lipid transfer activity for fluorescently labeled phosphatidylcholine, but no cryoprotective activity. Conversely, cryoprotectin showed no lipid transfer activity. It can be concluded that these structurally very similar proteins are functionally different in vitro and may also play distinct roles in plant metabolism.

Abstract 1998